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Self-supplied tumor oxygenation through separated liposomal delivery of H2O2 and catalase for enhanced radio-immunotherapy of cancer Xuejiao Song, Jun Xu, Chao Liang, Yu Chao, Qiutong Jin, Chao Wang, Meiwan Chen, and Zhuang Liu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b02720 • Publication Date (Web): 24 Sep 2018 Downloaded from http://pubs.acs.org on September 24, 2018

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Self-supplied tumor oxygenation through separated liposomal delivery of H2O2 and catalase for enhanced radio-immunotherapy of cancer Xuejiao Song1,2, Jun Xu2, Chao Liang2, Yu Chao2, Qiutong Jin2, Chao Wang2, Meiwan Chen1*, Zhuang Liu2*

1, State Key Laboratory of Quality Research in Chinese Medicine Institute of Chinese Medical Sciences University of Macau, Macau 999078 , China 2, Institute of Functional Nano & Soft Materials (FUNSOM) & Collaborative Innovation Center of Suzhou Nano Science and Technology Soochow University, Suzhou 215123, China E-mail: [email protected] E-mail: [email protected]

Abstract The recent years have witnessed the blooming of cancer immunotherapy, as well as their combinational use together with other existing cancer treatment techniques including radiotherapy. However, hypoxia is one of several causes of the immunosuppressive TME. Herein, we develop an innovative strategy to relieve tumor hypoxia by delivering exogenous H2O2 into tumors and the subsequent catalase-triggered H2O2 decomposition. In our experiment, H2O2 and catalase are separately loaded within stealthy liposomes. After intravenous (i.v.) pre-injection of CAT@liposome, another dose of H2O2@liposome is injected 4 h later. The sustainably released H2O2 could be decomposed by CAT@liposome, resulting in a long lasting effect in tumor oxygenation enhancement. As the result, the combination treatment by CAT@liposome plus H2O2@liposome offers remarkably enhanced therapeutic effects in cancer radiotherapy as observed in a mouse tumor model as well as a more clinically relevant patient-derived xenograft tumor model. Moreover, the relieved tumor hypoxia would reverse the immunosuppressive TME to favor anti-tumor immunities, further enhancing the combined radio-immunotherapy with cytotoxic T lymphocyte-associated antigen 4 (CTLA4) blockade. This work presents a simple yet effective strategy to promote tumor oxygenation via sequential delivering catalase and exogenous H2O2 into tumors using well-established liposomal carriers, showing great potential for clinical translation in radio-immunotherapy of cancer. 1

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Key words: Tumor hypoxia, self-supplied oxygen delivery, radiotherapy, check-point-blockade immunotherapy

Radiation therapy (RT), which has been extensively used in clinic cancer treatment, kills cancer cells primarily by generating DNA double strand breaks under high-energy ionization radiation rays (e.g. X-ray or γ-ray)1, 2. On the other hand, it has been well-recognized that radiation applied on tumors may be able to trigger anti-tumor immune responses by increasing antigen exposure on the surface of tumor cells3, 4. In the process of RT, the induced DNA damages and reactive oxygen species by radiation have been reported to result in inflammatory tumor cell death and release of damage associated molecular patterns, which can activate antigen presenting cells5, 6. Moreover, RT has also been shown to influence expression of cytokines and chemokines, which play critical roles in modulating immune responses7, 8. In general, RT would make cancer cells more sensitive to immune mediated attacks due to the above effects. Therefore, potential synergistic benefits may be achieved by combining RT with various immunotherapeutic treatment strategies. The tumor microenvironment (TME) is a complex system composed of not only tumor cells but also stromal cells, inflammatory cells, vasculature, and extracellular matrices (ECM). Owing to the insufficient blood flow and hyperpermeable vessels, TME is often characterized by hypoxia, acidity, and high interstitial fluid pressures9,

10

. It has been recognized that such tumor

microenvironment tends to be immunosuppressive, and would attenuate the immunotherapeutic efficacy via different mechanisms11-13.

Among these characteristics, hypoxia, as a hallmark of

tumors, has been reported to promote the polarization of immune-supportive M1-type tumor-associated macrophages (TAMs) to immunosuppressive M2-type TAMs, which are able to protect tumor cells from being attacked by the immune system14-16. On the other hand, oxygen is pivotal in preventing radiation-induced DNA damages from being restored by tumor cells 17. As the results within the hypoxic regions inside many types of solid tumors, RT-induced DNA damages and tumor cell killing would be much less significant18,

19

. Therefore, modulating the unfavorable

characteristics of TME, especially the tumor hypoxia, would be of great clinical value in improving both radiotherapy and immunotherapy of cancer20-23. Recently, many groups including ours have developed a number of different strategies to improve tumor oxygenation so as to reverse the hypoxia-associated resistance of tumors to various types of 2


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therapies24, 25. It has been reported that the tumor hypoxia status could be significantly relieved by increasing the intratumoral blood flow via methods such as normalization of tumor vasculatures26, 27, or simply by a mild hyperthermia effect locally applied on the tumors28. Artificial blood substitutes such as perfluorocarbon have also been explored to improve the delivery of oxygen into solid tumors29, 30. On the other hand, taking advantage of excessive amounts of hydrogen peroxide (H2O2) within the tumor microenvironment, several different groups including ours have uncovered that certain types of catalysts such as MnO2 nanostructures or catalase enzyme could induce the decomposition of tumor endogenous H2O2 and generate oxygen in situ31-34. Despite encouraging results achieved in those previous studies, there are still limitations for the abovementioned strategies to enhance tumor oxygenation. For delivering of oxygen into tumors via blood flow, its efficiency is often limited by the oxygen carrying ability of hemoglobin or PFC, as well as the blood vessel densities within the tumor. For in situ generation of oxygen within the tumor, however, the amount of endogenous H2O2 is limited (10~50 µM)35, 36 and would be variable between different types of solid tumors. Therefore, we propose another strategy for highly effective tumor oxygenation by delivering exogenous H2O2 into tumors and the subsequent catalase-triggered H2O2 decomposition / oxygen generation. As the maximal concentration of H2O2 within the aqueous phase (30% by weight) could be 5.8 x 102 times or 1.1 x 102 times higher than the solubility of oxygen molecules in blood or PFC, it is predicted that such a method may offer a highly effective approach for tumor oxygenation. In this contribution, polyethylene glycol (PEG) modified ‘stealthy liposomes’ are employed to separately load either catalase (CAT@liposome) or H2O2 (H2O2@liposome). H2O2, as a less-polar molecule, has been reported to be able to gradually diffuse through the lipid bilayer of liposomes37, 38, acting as the ‘fuel’ for CAT@liposome to produce oxygen. Utilizing the passive tumor homing ability of liposomal carriers, sequential intravenous (i.v.) injection of CAT@liposome and then H2O2@liposome (at 4 h later) could lead to significantly enhanced tumor oxygenation. Compared with single CAT@liposome injection to decompose only the tumor endogenous H2O2, utilizing the nano-supplier of H2O2 in the group of CAT@liposome plus H2O2@liposome injection offers a remarkably enhanced and long-lasting effect in relieving tumor hypoxia throughout the whole tumor. As the result, greatly improved cancer radiotherapy efficacies are achieved by this method in a mouse tumor model as well as a more clinically relevant patient-derived xenograft tumor model. Meanwhile, the relieved tumor hypoxia would result in the reversed immunosuppressive TME to 3


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favor anti-tumor immunities. With further combination use of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) checkpoint blockade, the enhanced RT treatment could promote infiltration of cytotoxic T lymphocytes (CTLs) and further inhibit tumor growth in the radio-immunotherapy of cancer after tumor hypoxia relief by our approach. Finally, owing to the pre-injection of CAT@liposome to eliminate excess H2O2 delivered by H2O2@liposome, such a treatment protocol renders no appreciable toxic effect to the treated animals. Therefore, our work presents an innovative yet simple tumor oxygenation strategy via delivering exogenous H2O2 into tumors using a well-established liposomal carrier, showing great potential for clinical translation. Stealth PEGylated liposomes have been extensively explored in recent decades as nanoscale carriers for various therapeutic or imaging agents owing to their excellent biocompatibility, long blood circulation time and versatile loading capacities for both hydrophilic and hydrophobic cargos39-43. In this work, we utilized a classical method to fabricate PEGylated liposomes to encapsulate either hydrogen peroxide (H2O2@Liposome), or catalase (CAT@Liposome), within their hydrophilic cavities (Figure 1a). As shown by transmission electron microscopy (TEM) images, both H2O2@Liposome and CAT@Liposome exhibited uniform size distributions (Figure S1a). The average diameters of the liposome nanoparticles were determined to be approximately 140 nm by dynamic light scattering (DLS) (Figure S1b), in accordance with TEM images. After removal of excess H2O2 or CAT by using Sephacryl S300 column, the loading efficiencies of H2O2 and CAT were calculated to be about (9.1+1.9) % and (12.8+1.2) %. We further investigated the release profile of H2O2. It was found that H2O2@Liposome exhibited a sustained release of H2O2 (Figure 1b), with 26% of total H2O2 released within 24 h. Meanwhile, the enzymatic activity changes of free catalase and CAT@liposome under protease K digestion were studied. It was found that CAT encapsulated within liposome could maintain 83% of its initial catalytic activity after being treated with protease K for 12 h, while free CAT lost the majority of its catalytic activity after protein K treatment, suggesting that the liposomal encapsulation could protect CAT from protease-mediated degradation (Figure S1c). Notably, the instant small drop of CAT catalytic activity for the CAT@liposome sample right after addition of protease K might be due to the possibility that a small fraction of enzyme molecules were adherent to the outside surface of liposomes. Furthermore, the oxygen generation rates of free H2O2 or H2O2@Liposome solution in the presence of CAT@Liposome was also studied (Figure 1c & Figure S1d). When free H2O2 was 4


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added into CAT@Liposome solution, a burst-like generation of oxygen was observed, and then the oxygen concentration sharply decreased due to the rapid exhaustion of H2O2. On the contrary, for the mixture of CAT@Liposome and H2O2@Liposome, we observed a moderate increase of oxygen concentrations, which could be attributed by the constant release of H2O2 from H2O2@Liposome to supply ‘fuel’ for catalase within CAT@Liposome. Next, we tested the potential toxicity of free H2O2, CAT@Liposome or H2O2@Liposome to 4T1 murine breast cancer cells by standard MTT assays as well as crystal violet staining assays. The relative viabilities of cells after being incubated with different agents for 24 h were determined. No appreciable negative effect to the cell viability of CAT@Liposome was observed even at high concentrations (Figure 2a & Figure S2a). On the other hand, H2O2, as a kind of reactive oxygen species (ROS), has been reported to result in apoptosis at moderate doses and necrosis at high doses. As expected, free H2O2 showed significant cell killing effect even at low concentrations (0.4 mM) (Figure 2b & Figure S2b), while H2O2@Liposome exhibited obviously attenuated cytotoxicity. However, the cell viability was still dramatically decreased at high concentrations of H2O2@Liposome (Figure 2c & Figure S2c). Interestingly, when the cancer cells were firstly incubated with non-toxic CAT@Liposome for 4 h followed with H2O2@Liposome incubation for another 20 h, no significant decrease in cell viabilities was observed even at high concentrations of H2O2@Liposome, demonstrating that the cytotoxicity of H2O2 could be effectively eliminated after decomposition by CAT@Liposome (Figure 2d & Figure S2d). Furthermore, reactive oxygen species (ROS)-indicator, 2’,7’-dichlorofluorescin-diacetate (DCFH-DA) was applied to detect the intracellular ROS levels under different treatments. As expected, high levels of ROS could be observed in the cells treated with H2O2@Liposome, which might due to the intracellular delivery and release of H2O2. Remarkably, the DCF signals were significantly decreased to the background level when the cells were incubated with non-toxic CAT@Liposome and H2O2@Liposome, indicating the released H2O2 could be rapidly decomposed by CAT@Liposome (Figure 2e & f). Next, we investigated in vivo behaviors of H2O2@Liposome and CAT@Liposome. 1,1’-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR), a commercial lipophilic NIR dye, was utilized here to label H2O2@Liposome and CAT@Liposome by inserting into the lipid bilayer structure of liposomes44, 45. The obtained DiR-H2O2@Liposome and DiR-CAT@Liposome nanoparticles (200 µL, containing 0.6 mg/mL DiR) were intravenously (i.v.) injected to the 4T1 5


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tumor bearing mice. Blood samples were collected at various time points and lysed for fluorescence measurement to determine the blood circulation profiles of those liposome nanoparticles. It was found that both DiR-H2O2@Liposome and DiR-CAT@Liposome nanoparticles exhibited long blood circulation time, with half-lives measured to be 12.86 h and 11.77 h, respectively (Figure 3a). At 24 h post injection, those tumor-bearing mice were sacrificed with major organs or tissues collected for ex vivo imaging by a Maestro EX in vivo fluorescence imaging system (CRi, Inc.) (Figure 3b). Strong DiR fluorescence signals in the tumor were observed for mice injected with both DiR-H2O2@Liposome and DiR-CAT@Liposome, demonstrating the high tumor uptake of these liposome nanoparticles via the enhanced permeability and retention (EPR) effect (Figure 3c). Moreover, we further investigated the catalase activity in blood at different time after injection according to the chromogenic assay. It was found that catalase in the blood after i.v injection could maintain ~60% of its initial catalytic activity at 24 h post injection (p.i.) (Figure S3a). Meanwhile, catalase activity in the tumor after i.v injection was also investigated. Tumors were obtained 24 h after i.v injection with CAT@Liposome and then homogenized. It was found the tumor homogenate from mice injected with CAT@Liposome could trigger rapid decomposition of H2O2 to generate a large amount of oxygen, compared with the tumor homogenate from untreated mice (Figure S3b). The results indicated the CAT delivered to the tumor by Liposome could maintain a high activity even after 24 h. Considering the two liposome nanoparticles were i.v injected separately, we further investigated the intra-tumoral distribution of the two types of nanoparticles. CAT@Liposome were labeled with FITC, while H2O2@Liposome were labeled with DiD. H2O2@Liposome (DiD) were i.v injected at 4 h after injection of CAT@Liposome (FITC). After another 20 h, the mice were sacrificed and the tumors were obtained and sliced for immunofluorescence staining of blood vessels. Confocal fluorescence images of tumor slices showed that the two liposome nanoparticles were both distributed around the blood vessels, ensuring that the released H2O2 could be subsequently decomposed by the nearby CAT@Liposome to generate oxygen (Figure S4). Next, we investigated whether H2O2@Liposome and CAT@Liposome nanoparticles could relieve tumor hypoxia in vivo. Taking advantage of the different absorbance of oxygenated hemoglobin (λ= 850 nm) and deoxygenated hemoglobin (λ= 750 nm), in vivo photoacoustic (PA) imaging could be employed to monitor the oxygenated hemoglobin levels within tumors46. Therefore, 6


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4T1 tumor bearing were either i.v. injected with CAT@Liposome (dose of CAT = 1.5 mg/mL) alone, or i.v. injected with H2O2@Liposome (dose of H2O2 = 50 mM) at 4 h post pre-injection of CAT@Liposome at the same dose. PA imaging was conducted at different time points using a Vevo LAZR Imaging System (FujiFilm VisualSonics Inc.) (Figure 3d&e). The tumor oxygenation of both two groups were significantly increased in the first 4 h owing to the CAT@Liposome triggered decomposition of tumor endogenous H2O2 into O2. Interestingly, with H2O2@Liposome injection, the oxygenated hemoglobin signals further increased throughout the whole tumor area, to a level much higher than that with CAT@Liposome treatment alone. Thus, our exogenous H2O2 supplier, H2O2@Liposome, by delivering a large amount of oxygen precursor H2O2 into tumors, could effectively improve the tumor oxygenation together with CAT@Liposome. To further confirm the tumor hypoxia relieving ability by our unique approach of exogenous H2O2

delivery,

pimonidazole,

a

hypoxia

probe,

was

utilized

for

hypoxia-specific

immunofluorescence staining assay47. Mice bearing 4T1 tumors were i.v. injected with phosphate buffered

saline

(PBS),

H2O2@Liposome,

CAT@Liposome,

or

CAT@Liposome

plus

H2O2@Liposome (4 h later after CAT@Liposome injection), and scarified at 24 h post injection (p.i.) for tumor collection and staining. Large-area fluorescence imaging was conducted to examine the hypoxia status of entire tumors ((Figure 3f). Quantitative analysis of hypoxia-positive percentages was then carried out for more than 10 slices of each tumor (Figure 3g). For tumors on mice injected with PBS or H2O2@Liposome alone, large hypoxia positive areas at 61% or 63%, respectively, were observed. As expected, CAT@Liposome treatment could obviously relieve tumor hypoxia. However, the hypoxia positive area measured by semi-quantitative analysis was still as high as ~27% in this group. In contrast, for tumors treated by CAT@Liposome plus H2O2@Liposome, their hypoxia positive area decreased to a rather low level at ~9% (Figure 3f & g & Figure S5). Therefore, our immunofluorescence staining results, being consistent with the PA imaging data, further confirmed that the extra supply of H2O2 by H2O2@Liposome in combination with CAT@Liposome could offer remarkably improved tumor oxygenation throughout the whole tumor area, and would be obviously superior to CAT@Liposome treatment alone to only decompose tumor endogenous H2O2. Next, in vivo radiotherapy was carried out. 4T1 tumor bearing mice were randomly divided into 5 group (six mice each group) (Figure 4a): 1, control group with PBS injection (Control); 2, i.v injection of CAT@Liposome (200 µl, CAT = 1.5 mg/mL) plus the subsequent injection of 7


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H2O2@Liposome (200 µl, H2O2 = 50 mM) 4 h later (Both, X-ray -); 3, PBS injection plus X-ray radiation (X-ray +); 4, i.v injection of CAT@Liposome (CAT=1.5 mg/mL) and then irradiated by X-ray (CAT@Liposome, X-ray +); 5, subsequent i.v injection of CAT@Liposome plus H2O2@Liposome (4 h later), and X-ray radiation (Both, X-ray +). The X-ray radiation dose at 8 Gy was given at 24 h post injection of the first agent. Following specific treatments, the tumor volumes in different groups were recorded. Compared to the tumor growth in the radiotherapy alone group, i.v. injection of CAT@Liposome before X-ray radiation offered slightly improved therapeutic efficacy. More importantly, the strongest tumor suppression effect was observed for radiotherapy after subsequent co-treatment of mice with CAT@Liposome plus H2O2@Liposome prior to X-ray radiation of tumors, owing to the most effective tumor oxygenation achieved in this group (Figure 4b&c). Moreover, hematoxylin and eosin (H&E) staining assay for tumor slices collected at the second day post various treatments further evidenced that the X-ray induced tumor destruction was the most significant for tumors on mice treated with CAT@Liposome plus H2O2@Liposome (Figure 4d & Figure S6). In recent years, patient-derived xenograft (PDX) tumor models established by implanting live tumor specimen harvested directly from the patient into an immunodeficient mouse, has been recognized to be a better mimic of human tumors with greater clinical relevance compared to xenograft tumor models based on cell lines48, 49. Herein, we further carried out in vivo experiment on a PDX tumor model by implanting Balb/C nude mice with human prostatic tumor samples harvested from a patient donor, as hypoxia has been reported to be a common feature for prostate tumors (Figure 5a)18, 50, 51. In order to demonstrate our strategy can efficiently reverse the hypoxia for prostatic PDX tumors, in vivo PA imaging and ex vivo immunofluorescence staining assay were carried out. Similar to that observed with the 4T1 tumor model, for mice injected with H2O2@Liposome 4 h post i.v. injection of CAT@Liposome, their tumor oxygenation level showed further increase compared to those with CAT@Liposome injection alone. Due to the extra supply of H2O2 by H2O2@Liposome, the oxyhemoglobin saturation levels over the entire tumor area (sO2 Avr Tot) increased from 5.9% to 26%, while tumor oxygenation levels of mice injected single CAT@Liposome only increased from 5.3% to 19% (Figure 5b&c). Consistently, the immunofluorescence staining assay with the hypoxia probe (pimonidazole) further confirmed that the tumor hypoxia status showed dramatic decrease for 8


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mice with subsequent treatment of CAT@Liposome plus H2O2@Liposome, whereas the tumor hypoxia relieving function of CAT@Liposome treatment alone was less significant (Figure S7a&b). In vivo radiotherapy was further conducted on PDX tumor bearing mice, which were randomly divided into five groups for different treatments: 1, Control; 2, CAT@Liposome plus H2O2@Liposome (Both), X-ray -; 3, X-ray +; 4, CAT@Liposome, X-ray +; 5, CAT@Liposome plus H2O2@Liposome (Both), X-ray +. In group 2 and 5, H2O2@Liposome was i.v. injected into mice at 4 h post i.v. injection of CAT@Liposome. All above treatments were conducted following exact the same parameters and doses as we used for the treatment of 4T1 tumor model. As expected, owing to the long lasting effect in tumor oxygenation enhancement by subsequent injection of CAT@Liposome and H2O2@Liposome, we achieved greatly improved cancer radiotherapy efficacy to treat PDX tumors in group 5, in which the tumor growth inhibition effect was the most significant compared to radiotherapy alone (group 3) or radiotherapy enhanced by CAT@Liposome alone (group 4) (Figure 5d&e). With the rapid development of cancer immunotherapy, the combination use of radiotherapy together with check point blockade strategies has already been shown positive results in a number of clinical tests52,

53

. However, in recent years, accumulated evidences have also shown that the

heterogeneity of tumors and the hostile TME features such as hypoxia could lead to the limited therapeutic outcomes in cancer immunotherapy12,

16

. Considering the long lasting effect of our

strategy in relieving tumor hypoxia, we thus studied whether Liposome@CAT plus Liposome@H2O2 treatment could regulate the phenotype of tumor associated macrophages (TAMs). As revealed by Figure 6a&b, an obvious reduction of M2-polarized TAMs (CD206hiCD11b+F4/80+) was observed after i.v injection of Liposome@CAT plus Liposome@H2O2, to a level that was notably lower than that achieved by i.v injection of Liposome@CAT alone. In contrast, no significant change of TAMs phenotypes was observed in the tumor after Liposome@H2O2 treatment, indicating that the reduction of M2-polarized TAMs was contributed by the efficiently relieved tumor hypoxia. Meanwhile, the secretion of IL-10 (predominant cytokine secreted by M2 macrophages) in the supernatant of tumor lysates decreased by 2.22 times for the Liposome@CAT plus Liposome@H2O2 treated mice (Figure 6c), and the secretion of IL-12 (predominant cytokine secreted by M1 macrophages) in the tumor exhibited significant upregulation (Figure 6d). All these results demonstrated that our strategy could effectively reverse the immunosuppressive TME by relieving tumor hypoxia. 9


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Thus, we wonder whether combined radiotherapy and immunotherapy can achieve more significant efficacy in tumor growth inhibition, after the tumor hypoxia is relieved by our strategy (Figure 6e). The blockade of CTLA4, an important negative T-cell stimulatory receptor, has been approved by the U.S. Food and Drug Administration (FDA) for treatment of several different types of cancer 54-56. In our experiment, CT26 tumor bearing mice were randomly divided into six group (5 mice each group): 1, control group with PBS injection (Control); 2, i.v injection of anti-CTLA-4 (α-CTLA-4); 3, X-ray radiation (X-ray); 4, X-ray radiation plus i.v injection of anti-CTLA-4 (X-ray, α-CTLA-4); 5, subsequent i.v injection of CAT@Liposome plus H2O2@Liposome (4 h later), and with X-ray radiation (Both, X-ray); 6, subsequent i.v injection of CAT@Liposome plus H2O2@Liposome (4 h later), with X-ray radiation, and i.v injection of anti-CTLA-4 (Both, X-ray, α-CTLA-4). Anti-CTLA-4 antibody at a dose of 500 µg kg−1 was given by i.v. injection at day 1, 3, 5, and 7 post X-ray irradiation in group 2, 4 and 6. Tumor sizes were recorded every 2 days (Figure 6f). It was found that the therapeutic benefit of anti-CTLA-4 treatment was not significant if it was used alone (Group 2), or in combination with conventional radiotherapy (Group 4), compared to the respective counterpart control groups, Group 1 and Group 3, respectively. Such a phenomenon may be attributed to the immune-suppressive TME that limits the efficacy of checkpoint blockade therapy. In marked contrast, for mice treated with Liposome@CAT plus Liposome@H2O2 to promote their tumor oxygenation, obviously enhanced efficacy was achieved in the radio-immunotherapy with X-ray

plus

anti-CTLA-4

(Group

6),

compared

to

that

achieved

by

conventional

radio-immunotherapy (Group 4), or oxygenation-enhanced radiotherapy alone (Group 5) (Figure 6f-h). Moreover, during the therapy, there was no significant decrease in the mouse body weights (Figure S8). To understand such phenomenon, we carefully studied the mechanisms of antitumor immune responses during combined radiotherapy-immunotherapy after tumor hypoxia relief by our strategy. Cytotoxic T lymphocytes (CTLs, CD3+CD4-CD8+) that could directly kill cancer cells play the critical role in cancer immunotherapy. Therefore, the tumors of each group after various treatments were collected for immune cells analysis on day 7. It was found that the percentages of CD8+ CTLs in the tumors of mice after radiotherapy with Liposome@CAT plus Liposome@H2O2 showed was obviously increased compared to other groups, and anti-CTLA-4 antibody treatment could further remarkably enhance the CTL infiltration (Figure 6i & Figure S9a&b). On the other hand, it was 10


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found that after introduction of α-CTLA-4 antibody, the percentage of regulatory T cells (Tregs) (CD3+CD4+Foxp3+) in the tumor showed significant decrease, indicating that CTLA-4 blockade was able to effectively abrogate the activity of Tregs (Figure 6j & Figure S9c&d). We also compared the ratio of CD8+ T cells to Tregs (Figure 6k). It could be observed that the ratio of CD8+ T cells to Tregs in the tumors from Group 6 (Liposome@CAT plus Liposome@H2O2, X-ray, together with α-CTLA-4) remarkably increased. All the above results indicated that RT treatment after subsequent injection of Liposome@CAT plus Liposome@H2O2 combining with α-CTLA-4 antibody could synergistically induce highly effective antitumor immune responses to remarkably destruct tumors. As summarized in Figure 6l, owing to the long lasting effect in tumor hypoxia relief by subsequent injection of Liposome@CAT plus Liposome@H2O2, while the efficacy of RT could be significantly enhanced, the immunosuppressive TME would also be modulated to favor anti-tumor immunities (e.g. polarization of M2-type TAMs to the M1-type). When further combined with the use of CTLA-4 checkpoint blockade, the infiltration of CTLs would be promoted whereas the immune-suppressive Tregs could be restrained. As the results of those effects acting together, a remarkable synergistic therapeutic outcome in destructing tumors is achieved by the combined radio-immunotherapy of cancer after tumor hypoxia relief by our developed strategy. Considering the toxicity of H2O2, we at last carefully investigated the potential in vivo side effect of such treatment strategy. Mice were sacrificed at 1, 3 and 7 days after sequential injection with CAT@Liposome and H2O2@Liposome to obtain the major organs for H&E staining (Figure S10). Despite the high reticuloendothelial system (RES) uptake of the liposome nanoparticles in liver and spleen, no noticeable organ damage or inflammatory lesion was observed in all major mouse organs, most likely due to the rapid decomposition of retained H2O2 by pre-existing CAT@Liposome. Furthermore, the standard serum biochemistry assay and complete blood panel test were also carried out at 1, 3 and 7 days post injection of CAT@Liposome and H2O2@Liposome (Figure 7a-l). No significant difference of all measured parameters were found, demonstrating that injecting CAT@Liposome and H2O2@Liposome in sequence could be a safe treatment method for animals without inducing any appreciable toxicity or organ injury. In summary, we uncover in this work that subsequent systemic injection of two types of PEGlyated liposomes with H2O2 and catalase encapsulated would be an effective and safe approach to dramatically improve the tumor oxygenation. With additional supply of H2O2, our method would 11


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be more effective than the in-situ tumor oxygenation strategy by only decomposing the limited amount of tumor endogenous H2O2. Compared to delivering oxygen into tumors with oxygen carriers (e.g. PFC, hemoglobin nanoparticles), delivering H2O2 as an oxygen precursor would be more efficient considering the much higher solubility of H2O2 (several orders of magnitude higher than the solubility of oxygen molecules even within carriers). As the results of the greatly enhanced tumor oxygenation by the subsequent injection of CAT@Liposome and H2O2@Liposome, the tumor suppression efficacy by radiotherapy could be remarkably enhanced, as demonstrated in a cell-line-based mouse 4T1 tumor model as well as a more clinically relevant human prostatic PDX tumor model. Moreover, while the existence of CAT@Liposome would eliminate the in vitro cytotoxicity of H2O2@Liposome, subsequent injection of CAT@Liposome and H2O2@Liposome resulted in no appreciable in vivo toxicity as illustrated by our careful histology examinations and blood assays. Our work for the first time illustrates that by delivering H2O2, a well-known cytotoxic ROS, together with its decomposing enzyme into tumors using liposomal carriers, the therapeutic efficacy of clinically tested radio-immunotherapy could be greatly improved, without causing additional toxic side effects. Such a strategy would have great value in terms of future clinical translation, and may also be applied to treat other classes of diseases originated for oxygen deficient in certain organs (e.g. cardiovascular or chronic obstructive lung disease).

Acknowledgement This work was partially supported by the National Research Programs from Ministry of Science and Technology (MOST) of China (2016YFA0201200), the National Natural Science Foundation of China (51525203, 81403120, 21603155), Collaborative Innovation Center of Suzhou Nano Science and Technology, and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the Macao Science and Technology Development Fund (096/2015/A3) and the University of Macau (MYRG2016-00130-ICMS-QRCM).

Supporting Information Available: Synthetic and experimental details, additional information including TEM images, DLS profiles, enzymatic activities, in vitro cytotoxicity data, catalase activities in blood and tumor, the distribution of nanoparticles in the tumor, full-scale 12


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immunofluorescence images, immunofluorescence images of PDX tumor slices, body weight data for mice after different treatments, the flow cytometry gating strategies, and micrographs of H&E stained organ slices.

Reference 1.

Mansfield, C. M.; Komamicky, L. T.; Reisinger, S.; Rosenberg, A. L.; Krishnan, L.; Jewell, W. R.; Benammar, A.; Taylor, J.

Cancer Detect Prev 1994, 18, (6), 493-499. 2.

Qiao, X. Y.; Tullgren, O.; Lax, I.; Sirzen, F.; Lewensohn, R. Lung Cancer-J Iaslc 2003, 41, (1), 1-11.

3.

Teng, F. F.; Kong, L.; Meng, X. J.; Yang, J.; Yu, J. M. Cancer Letters 2015, 365, (1), 23-29.

4.

Twyman-Saint Victor, C.; Rech, A. J.; Maity, A.; Rengan, R.; Pauken, K. E.; Stelekati, E.; Benci, J. L.; Xu, B. H.; Dada, H.;

Odorizzi, P. M.; Herati, R. S.; Mansfield, K. D.; Patsch, D.; Amaravadi, R. K.; Schuchter, L. M.; Ishwaran, H.; Mick, R.; Pryma, D. A.; Xu, X. W.; Feldman, M. D.; Gangadhar, T. C.; Hahn, S. M.; Wherry, E. J.; Vonderheide, R. H.; Minn, A. J. Nature 2015, 520, (7547), 373-377. 5.

Derer, A.; Frey, B.; Fietkau, R.; Gaipl, U. S. Cancer Immunol Immun 2016, 65, (7), 779-786.

6.

Sharabi, A. B.; Lim, M.; DeWeese, T. L.; Drake, C. G. Lancet Oncol 2015, 16, (13), E498-E509.

7.

Burnette, B. C.; Liang, H.; Lee, Y.; Chlewicki, L.; Khodarev, N. N.; Weichselbaum, R. R.; Fu, Y. X.; Auh, S. L. Cancer

Research 2011, 71, (7), 2488-2496. 8.

Ishihara, H.; Tsuneoka, K.; Dimchev, A. B.; Shikita, M. Radiation Research 1993, 133, (3), 321-326.

9.

Hockel, M.; Vaupel, P. Journal of the National Cancer Institute 2001, 93, (4), 266-276.

10. Finger, E. C.; Giaccia, A. J. Cancer Metast Rev 2010, 29, (2), 285-293. 11. Leibovici, J.; Itzhaki, O.; Huszar, M.; Sinai, J. Immunotherapy-Uk 2011, 3, (11), 1385-1408. 12. Tang, H. D.; Qiao, J.; Fu, Y. X. Cancer Letters 2016, 370, (1), 85-90. 13. Riaz, N.; Havel, J. J.; Makarov, V.; Desrichard, A.; Urba, W. J.; Sims, J. S.; Hodi, F. S.; Martin-Algarra, S.; Mandal, R.; Sharfman, W. H.; Bhatia, S.; Hwu, W. J.; Gajewski, T. F.; Slingluff, C. L.; Chowell, D.; Kendall, S. M.; Chang, H.; Shah, R.; Kuo, F. S.; Morris, L. G. T.; Sidhom, J. W.; Schneck, J. P.; Horak, C. E.; Weinhold, N.; Chan, T. A. Cell 2017, 171, (4), 934-949. 14. Chanmee, T.; Ontong, P.; Konno, K.; Itano, N. Cancers 2014, 6, (3), 1670-1690. 15. Noy, R.; Pollard, J. W. Immunity 2014, 41, (5), 866-866. 16. Henze, A. T.; Mazzone, M. J Clin Invest 2016, 126, (10), 3672-3679. 17. Brown, J. M.; William, W. R. Nature Reviews Cancer 2004, 4, (6), 437-447. 18. Milosevic, M.; Warde, P.; Menard, C.; Chung, P.; Toi, A.; Ishkanian, A.; McLean, M.; Pintilie, M.; Sykes, J.; Gospodarowicz, M.; Catton, C.; Hill, R. P.; Bristow, R. Clinical Cancer Research 2012, 18, (7), 2108-2114. 19. Zips, D.; Adam, M.; Flentje, M.; Haase, A.; Molls, M.; Mueller-Klieser, W.; Petersen, C.; Philbrook, C.; Schmitt, P.; Thews, O.; Walenta, S.; Baumann, M. Strahlenther Onkol 2004, 180, (10), 609-615. 20. Koukourakis, M. I.; Giatromanolaki, A.; Skarlatos, J.; Corti, L.; Blandamura, S.; Piazza, M.; Gatter, K. C.; Harris, A. L. Cancer Research 2001, 61, (5), 1830-1832. 21. Liu, Y. Y.; Liu, Y.; Bu, W. B.; Cheng, C.; Zuo, C. J.; Xiao, Q. F.; Sun, Y.; Ni, D. L.; Zhang, C.; Liu, J. A.; Shi, J. L. Angew Chem Int Edit 2015, 54, (28), 8105-8109. 22. Huang, C. C.; Chia, W. T.; Chung, M. F.; Lin, K. J.; Hsiao, C. W.; Jin, C.; Lim, W. H.; Chen, C. C.; Sung, H. W. Journal of the American Chemical Society 2016, 138, (16), 5222-5225. 23. Qian, C. G.; Yu, J. C.; Chen, Y. L.; Hu, Q. Y.; Xiao, X. Z.; Sun, W. J.; Wang, C.; Feng, P. J.; Shen, Q. D.; Gu, Z. Advanced 13


Nano Letters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 25

Materials 2016, 28, (17), 3313-3320. 24. Moeller, B. J.; Richardson, R. A.; Dewhirst, M. W. Cancer Metast Rev 2007, 26, (2), 241-248. 25. Song, X. J.; Feng, L. Z.; Liang, C.; Gao, M.; Song, G. S.; Liu, Z. Nano Research 2017, 10, (4), 1200-1212. 26. Jain, R. K. Science 2005, 307, (5706), 58-62. 27. Huang, Y. H.; Yuan, J. P.; Righi, E.; Duda, D. G.; Fukumura, D.; Poznanasky, M. C.; Jain, R. K. Cancer Research 2013, 73, (8). 28. Yi, X.; Yang, K.; Liang, C.; Zhong, X. Y.; Ning, P.; Song, G. S.; Wang, D. L.; Ge, C. C.; Chen, C. Y.; Chai, Z. F.; Liu, Z. Advanced Functional Materials 2015, 25, (29), 4689-4699. 29. Song, G. S.; Liang, C.; Yi, X.; Zhao, Q.; Cheng, L.; Yang, K.; Liu, Z. A. Advanced Materials 2016, 28, (14), 2716-2723. 30. Xu, L. F.; Qiu, X. F.; Zhang, Y. T.; Cao, K.; Zhao, X. Z.; Wu, J. H.; Hu, Y. Q.; Guo, H. Q. J Transl Med 2016, 14. 31. Zheng, D. W.; Li, B.; Li, C. X.; Fan, J. X.; Lei, Q.; Li, C.; Xu, Z. S.; Zhang, X. Z. Acs Nano 2016, 10, (9), 8715-8722. 32. Gordijo, C. R.; Abbasi, A. Z.; Amini, M. A.; Lip, H. Y.; Maeda, A.; Cai, P.; O'Brien, P. J.; DaCosta, R. S.; Rauth, A. M.; Wu, X. Y. Advanced Functional Materials 2015, 25, (12), 1858-1872. 33. Zhu, W. W.; Dong, Z. L.; Fu, T. T.; Liu, J. J.; Chen, Q.; Li, Y. G.; Zhu, R.; Xu, L. G.; Liu, Z. Advanced Functional Materials 2016, 26, (30), 5490-5498. 34. Song, G. S.; Chen, Y. Y.; Liang, C.; Yi, X.; Liu, J. J.; Sun, X. Q.; Shen, S. D.; Yang, K.; Liu, Z. Advanced Materials 2016, 28, (33), 7143-7148. 35. Kuang, Y. Y.; Baakrishnan, K.; Gandhi, V.; Peng, X. H. Journal of the American Chemical Society 2011, 133, (48), 19278-19281. 36. Szatrowski, T. P.; Nathan, C. F. Cancer Research 1991, 51, (3), 794-798. 37. Frimer, A. A.; Forman, A.; Borg, D. C. Israel J Chem 1983, 23, (4), 442-445. 38. Chen, Q.; Liang, C.; Sun, X. Q.; Chen, J. W.; Yang, Z. J.; Zhao, H.; Feng, L. Z.; Liu, Z. P Natl Acad Sci USA 2017, 114, (21), 5343-5348. 39. Allen, T. M.; Cullis, P. R. Advanced drug delivery reviews 2013, 65, (1), 36-48. 40. Lasic, D.; Martin, F.; Gabizon, A.; Huang, S.; Papahadjopoulos, D. Biochimica et Biophysica Acta (BBA)-Biomembranes 1991, 1070, (1), 187-192. 41. Samad, A.; Sultana, Y.; Aqil, M. Current drug delivery 2007, 4, (4), 297-305. 42. Torchilin, V. P. Nature reviews Drug discovery 2005, 4, (2), 145-160. 43. Luo, D. D.; Carter, K. A.; Razi, A.; Geng, J. M.; Shao, S.; Giraldo, D.; Sunar, U.; Ortega, J.; Lovell, J. F. Biomaterials 2016, 75, 193-202. 44. Eisenblatter, M.; Ehrchen, J.; Varga, G.; Sunderkotter, C.; Heindel, W.; Roth, J.; Bremer, C.; Wall, A. Journal of Nuclear Medicine 2009, 50, (10), 1676-1682. 45. Schroeder, T. Nat Methods 2011, 8, (4), S30-S35. 46. Li, C.; Wang, L. V. Physics in medicine and biology 2009, 54, (19), R59. 47. Westbury, C. B.; Pearson, A.; Nerurkar, A.; Reis-Filho, J. S.; Steele, D.; Peckitt, C.; Sharp, G.; Yarnold, J. R. Brit J Radiol 2007, 80, (959), 934-938. 48. Ricci, F.; Bizzaro, F.; Cesca, M.; Guffanti, F.; Ganzinelli, M.; Decio, A.; Ghilardi, C.; Perego, P.; Fruscio, R.; Buda, A.; Milani, R.; Ostano, P.; Chiorino, G.; Bani, M. R.; Damia, G.; Giavazzi, R. Cancer Research 2014, 74, (23), 6980-6990. 49. Tentler, J. J.; Tan, A. C.; Weekes, C. D.; Jimeno, A.; Leong, S.; Pitts, T. M.; Arcaroli, J. J.; Messersmith, W. A.; Eckhardt, S. G. Nature Reviews Clinical Oncology 2012, 9, (6), 338-350. 50. Milosevic, M.; Bristow, R. G.; Menard, C.; Chung, P. W.; Bayley, A.; McLean, M.; Tai, A.; Gospodarowicz, M. K.; Catton, C.; Warde, P. R. Journal of Clinical Oncology 2011, 29, (7). 51. Gomez, C. R.; Munz, J. M.; Kosari, F.; Karnes, J.; Cheville, J.; Ida, C.; Sebo, T.; Vasmatzis, G.; Vuk-Pavlovic, S. Cancer Research 2012, 72, (8). DOI: 10.1158/1538-7445. 14


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Nano Letters

52. Weichselbaum, R. R.; Liang, H.; Deng, L. F.; Fu, Y. X. Nature Reviews Clinical Oncology 2017, 14, (6), 365-379. 53. Reynders, K.; Illidge, T.; Siva, S.; Chang, J. Y.; De Ruysscher, D. Cancer Treat Rev 2015, 41, (6), 503-510. 54. Ueda, H.; Howson, J. M. M.; Esposito, L.; Heward, J.; Snook, H.; Chamberlain, G.; Rainbow, D. B.; Hunter, K. M. D.; Smith, A. N.; Di Genova, G.; Herr, M. H.; Dahlman, I.; Payne, F.; Smyth, D.; Lowe, C.; Twells, R. C. J.; Howlett, S.; Healy, B.; Nutland, S.; Rance, H. E.; Everett, V.; Smink, L. J.; Lam, A. C.; Cordell, H. J.; Walker, N. M.; Bordin, C.; Hulme, J.; Motzo, C.; Cucca, F.; Hess, J. F.; Metzker, M. L.; Rogers, J.; Gregory, S.; Allahabadia, A.; Nithiyananthan, R.; Tuomilehto-Wolf, E.; Tuomilehto, J.; Bingley, P.; Gillespie, K. M.; Undlien, D. E.; Ronningen, K. S.; Guja, C.; Ionescu-Tirgoviste, C.; Savage, D. A.; Maxwell, A. P.; Carson, D. J.; Patterson, C. C.; Franklyn, J. A.; Clayton, D. G.; Peterson, L. B.; Wicker, L. S.; Todd, J. A.; Gough, S. C. L. Nature 2003, 423, (6939), 506-511. 55. Pardoll, D. M. Nature Reviews Cancer 2012, 12, (4), 252-264. 56. Topalian, S. L.; Taube, J. M.; Anders, R. A.; Pardoll, D. M. Nature Reviews Cancer 2016, 16, (5), 275-287.

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Figure 1. Preparation and characterization of H2O2@Liposome and CAT@Liposome. (a) A schematic diagram showing the liposome compositions and oxygen generation process. (b) The release profile of H2O2 from H2O2@Liposome within PBS. (c) Time-dependent changes of dissolved oxygen concentrations in different groups including pure water, H2O2@Liposome, CAT@Liposome, CAT@Liposome plus H2O2@Liposome, and CAT@Liposome plus free H2O2.

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Figure 2. In vitro cytotoxicity assays. (a-c) Relative viabilities of 4T1 cells after incubation with CAT@Liposome, free H2O2 and H2O2@Liposome at various concentrations for 24 h. (d) Relative viabilities of 4T1 cells after incubation with CAT@Liposome for 4 h followed with further incubation with various concentrations of H2O2@Liposome for 20 h. (e) Confocal images of 4T1 cells treated with different liposomes and then stained using DCFH-DA (green) to evaluate intracellular ROS generation. (f) Flow cytometry data for intracellular ROS levels. The data are shown as mean ± standard deviation (SD).

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Figure 3. In vivo behaviors of liposomal nanoparticles and their tumor hypoxia relief ability. (a) Blood circulation profiles of DiR-H2O2@Liposome and DiR-Liposome@CAT measured by the DiR 19


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fluorescence in the blood samples at different time points post-injection. (b&c) Ex vivo fluorescence images (b) and quantified fluorescence signals (c) of the major organs/tissues taken at 24 h post-injection: liver (Li), spleen (Sp), kidney (Ki), heart (He), lung (Lu), stomach (St), intestine (In), and tumor (Tu). (d) Representative PA images of 4T1 tumors on mice at various time points post injection with CAT@Liposome (upper row) or CAT@Liposome plus H2O2@Liposome (bottom row). (e) Quantification of the oxyhemoglobin saturation in the total tumor area (sO2 Avr Total) in (d). (f) Full-scale immunofluorescence images of the entire tumors after different treatments. The cell nuclei, blood vessels and hypoxic areas were stained with DAPI (blue), anti-CD31 antibody (red), and anti-pimonidazole antibody (green), respectively. The strong signals on the tumor boundary were due to the autofluorescence. (g) Quantification of tumor hypoxia and blood vessel densities from the images shown in (f). For the co-treatment group, H2O2@Liposome was given 4 h post injection of CAT@Liposome. The data are shown as mean ± SD (n=3). P values: **P < 0.01,***P < 0.001, ANOVA.

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Figure 4. In vivo radiotherapy on 4T1 tumor model. (a) A scheme showing the therapeutic procedure. (b) Tumor growth curves of different groups of mice (five mice per group) after various treatments including: 1, control group with PBS injection (Control); 2, i.v injection of CAT@Liposome plus the subsequent injection of H2O2@Liposome 4 h later (Both, X-ray -); 3, PBS injection plus X-ray radiation (X-ray +); 4, i.v injection of CAT@Liposome and then irradiated by X-ray (CAT@Liposome, X-ray +); 5, subsequent i.v injection of CAT@Liposome plus H2O2@Liposome (4 h later), and X-ray radiation (Both, X-ray +). (c) Average tumor weights measured at day 16 after different treatments shown in (b). (d) H&E stained tumor slices from different groups collected 48 h after injection of the first agent (24 h post X-ray radiation in group 3, 4, 5). The data are shown as mean±SD (n=5). P values: *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA.

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Figure 5. In vivo radiotherapy on the PDX tumor model. (a) A scheme illustrating the process of setting up the patient-derived prostate tumor model and in vivo radiotherapy. (b) Representative PA images of tumors at various time points post injection with CAT@Liposome (upper row) or CAT@Liposome plus H2O2@Liposome (bottom row). (c) Quantification of the oxyhemoglobin saturation in the total tumor area (sO2 Avr Total) in (b). (d) Tumor growth curves of mice after various treatments (five mice per group) including: 1, Control; 2, Both, X-ray -; 3, X-ray +; 4, CAT@Liposome, X-ray +; 5, Both, X-ray +. The X-ray radiation dose at 8 Gy was given 24 h post injection of the first agent. (e) Average tumor weight at day 18 after different treatments shown in (d). The data are shown as mean ± SD. P values: *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA. 22


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Figure 6. Combined radio-immunotherapy after tumor hypoxia relief. (a&b) Representative flow cytometric plots (a) and the corresponding quantification (b) of M2-type macrophages (CD206+) among TAMs (CD11b+F4/80+). (c&d) The levels of IL-10 (c) and IL-12 (d) in the supernatant of 23


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tumor lysates after various treatments (n=4). (e) Schematic illustration the combined radio-immunotherapy after tumor hypoxia relief by subsequent treatments of CAT@Liposome plus H2O2@Liposome. (f) Tumors growth curves of different groups of mice after various treatments as indicated (five mice per group). (g) Photos of tumors after various treatments. (h) The average tumor weights after various treatments. (i) The percentage of CTLs infiltrated into tumors after various treatments. CD3+CD8+ cells were defined as CTLs. (j) The quantification shows percentages (gated on CD4+ cells) of CD4+FoxP3+ Treg cells in tumors after various treatments indicated. (k) The ratio of CD8+ T cells to Tregs. (l) The proposed mechanism of anti-tumor immune responses induced by the combined radiotherapy with anti-CTLA-4 immunotherapy, after tumor hypoxia relief by CAT@Liposome / H2O2@Liposome. The data are shown as mean ± SD. P values: *P < 0.05, **P < 0.01, ***P < 0.001, ANOVA.

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